The long-term goal of this research is to understand the molecular mechanisms that could lead to the emergence of highly pathogenic H5N1 influenza viruses that are efficiently transmitted among humans. Recently, direct avian-to-human transmission of these viruses has been documented multiple times. Currently, no vaccine to this subtype is commercially available and recent findings suggest that H5N1 viruses are becoming more adept at infecting people. The proposed research builds on the investigator's access to current H5N1 viruses and the availability of a highly efficient reverse genetics system that allows the influenza virus genome to be modified at will. Aim 1 seeks to identify the molecular features required for adaptation of avian H5N1 viruses in humans. Comprehensive sequence analyses of human and avian virus isolates revealed candidate amino acids for the adaptation of avian viruses to humans. The importance of these amino acids to the replicative ability of viruses will be tested in mammalian species by using mutant viruses generated with reverse genetics. Aim 2 investigates the virulence and transmissibility of reassortants between H5N1 viruses and currently circulating human strains in mammalian species including mice, ferrets, and nonhuman primates. These studies are expected to provide information essential for assessing the pandemic potential of such reassortants. Aim 3 assesses the role of the amino acid at position 627 of the influenza PB2 protein for adaptation to and spread among mammalian species. Evidence suggests that Lys at this position may promote pathogenicity in mammals, possibly by conferring a growth advantage in the upper respiratory tract, hence facilitating efficient virus spread among humans. Aim 4 determines the pathogenic potential of a recently isolated H5N1 virus resistant to oseltamivir, an effective inhibitor of influenza virus neuraminidase. Aim 5 elucidates the molecular mechanisms for the acquisition of high virulence of H5N1 viruses in ducks. While avian influenza viruses are usually maintained asymptomatically in ducks, some H5N1 viruses acquired the ability to kill ducks in 2002. Reassortants between nonpathogenic and pathogenic duck virus isolates are expected to yield new insights into the molecular mechanisms of influenza pathogenesis. Collectively, these studies will enable better preparedness for future outbreaks of highly pathogenic avian influenza viruses in humans.